Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading

The postcranial skeleton of modern Homo sapiens is relatively gracile compared with other hominoids and earlier hominins. This gracility predisposes contemporary humans to osteoporosis and increased fracture risk. Explanations for this gracility include reduced levels of physical activity, the dissipation of load through enlarged joint surfaces, and selection for systemic physiological characteristics that differentiate modern humans from other primates. This study considered the skeletal remains of four behaviorally diverse recent human populations and a large sample of extant primates to assess variation in trabecular bone structure in the human hip joint. Proximal femur trabecular bone structure was quantified from microCT data for 229 individuals from 31 extant primate taxa and 59 individuals from four distinct archaeological human populations representing sedentary agriculturalists and mobile foragers. Analyses of mass-corrected trabecular bone variables reveal that the forager populations had significantly higher bone volume fraction, thicker trabeculae, and consequently lower relative bone surface area compared with the two agriculturalist groups. There were no significant differences between the agriculturalist and forager populations for trabecular spacing, number, or degree of anisotropy. These results reveal a correspondence between human behavior and bone structure in the proximal femur, indicating that more highly mobile human populations have trabecular bone structure similar to what would be expected for wild nonhuman primates of the same body mass. These results strongly emphasize the importance of physical activity and exercise for bone health and the attenuation of age-related bone loss.Compared with other hominoids and extinct hominin species, more recent humans possess relatively gracile postcranial skeletons (1–9). One of the consequences of this gracility in contemporary humans is an increased fracture risk associated with age-related bone loss and osteoporosis [hip fractures alone are projected to reach 6.26 million per year globally by 2050 (10)] (11–15). The etiology of this relative gracility remains uncertain, and this uncertainty hinders the development of strategies for mitigating fracture risk and morbidity. The progressive gracilization of the Homo postcranial skeleton was originally detected in cortical bone structure (1, 2), but has now been demonstrated in the trabecular bone microstructure of joints (12, 14, 16–19), where osteoporotic fracture risk is highest (20). Most notably, in an analysis of thoracic vertebral bodies, Cotter et al. (12) found that young adult humans have significantly lower trabecular bone volume fraction (BV/TV) and thinner vertebral shells than similarly sized apes. Griffin et al. (16) also found significantly lower BV/TV in the human first and second metatarsal heads compared with hominoid primates. The results of these studies are corroborated by work on the hominoid calcaneus (17), the anthropoid proximal femur (18), and several other clinical studies of femoral head trabecular bone architecture in contemporary adult humans (21–24). The results of these studies suggest that relative trabecular bone volume in the axial skeleton and lower limbs is significantly lower in modern humans compared with quadrupeds, despite the legs and vertebral column bearing a higher proportion of body mass and peak substrate reaction forces during bipedal locomotion (25–27). The high positive correlation between BV/TV and bone material properties (28–32) suggests that trabecular bone in the human lower limb and vertebral column is less stiff than in other primates. Ongoing debates aimed at enhancing our understanding of bone adaptation, skeletal health, and the prevention and treatment of osteoporosis would be greatly enhanced by the determination of the primary factors underlying the relatively gracile skeleton of living humans.Several explanations for the skeletal gracility of recent modern humans have been offered. The most common explanation is that living populations are simply less physically active compared with extinct hominins or closely related contemporary wild apes (1–6). This hypothesis suggests that a shift in subsistence patterns away from hunting and gathering, combined with an increased reliance on technology, led to reductions in overall physical activity levels and mobility in more recent hominins (cf. 2). In contrast, some (12, 13) attribute lower trabecular bone volume in humans to the mechanical consequences of the larger vertebral cross-sectional areas and larger joint surface areas required of an obligate biped. The crux of this argument is that larger joint surfaces distribute loads across a greater area, thereby reducing strain in the underlying trabecular tissue and leading to lower bone volume. Cotter et al. (12) have suggested that even if human activity levels were equal to those of wild apes, this loading would still be insufficient to illicit comparable trabecular bone growth. Alternatively, it has been suggested that the low bone-volume fraction observed in human thoracic vertebrae and the first and second metatarsals are the result of systemic physiological differences between humans and apes (14, 16). These studies do not suggest the mechanism or the function of this systemic gracility, but one potential explanation may be selection for increased tissue economy in hominins (5, 33–35).The aim of this study is to assess explanations for the trabecular bone gracility found in contemporary populations by evaluating the skeletal structure of human groups with divergent behavioral patterns within the broader context of primate biology. The impact of this research is twofold: (i) the samples and methods (imaging of trabecular bone microstructure using microCT) allow us to address questions inaccessible to research focused on living participants, yet inform on prevention and treatment in the 21st century; and (ii) these novel analyses allow us to evaluate the efficacy of quantifying trabecular bone structure for the purpose of differentiating activity and mobility patterns among prehistoric hominins. For this assessment, trabecular bone architecture of the proximal femur is compared among human foragers, village agriculturalists, and a large sample of extant primates. One of three outcomes is possible: (i) trabecular bone architecture does not separate Homo sapiens from the general nonhuman primate pattern, indicating a high level of canalization in primates for specific trabecular architectural features; (ii) trabecular bone architecture separates all H. sapiens from the nonhuman primate allometric pattern, indicating that trabecular bone structure in humans is largely driven by postcranial joint size or a genetic predisposition to maintaining a relatively gracile skeleton; or (iii) trabecular bone architecture separates agriculturalists and foragers, indicating structural differences among the groups, highlighting the influence of biomechanical loading or other osteogenic factors on trabecular bone composition.     

Estrogen receptor-α in osteocytes is important for trabecular bone formation in male mice

The bone-sparing effect of estrogen in both males and females is primarily mediated via estrogen receptor-α (ERα), encoded by the Esr1 gene. ERα in osteoclasts is crucial for the trabecular bone-sparing effect of estrogen in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both genders. We hypothesized that ERα in osteocytes is important for trabecular bone in male mice and for cortical bone in both males and females. Dmp1-Cre mice were crossed with ERα^flox/flox mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα^−/−). Male Dmp1-ERα^−/− mice displayed a substantial reduction in trabecular bone volume (−20%, P < 0.01) compared with controls. Dynamic histomorphometry revealed reduced bone formation rate (−45%, P < 0.01) but the number of osteoclasts per bone surface was unaffected in the male Dmp1-ERα^−/− mice. The male Dmp1-ERα^−/− mice had reduced expression of several osteoblast/osteocyte markers in bone, including Runx2, Sp7, and Dmp1 (P < 0.05). Gonadal intact Dmp1-ERα^−/− female mice had no significant reduction in trabecular bone volume but ovariectomized Dmp1-ERα^−/− female mice displayed an attenuated trabecular bone response to supraphysiological E2 treatment. Dmp1-ERα^−/− mice of both genders had unaffected cortical bone. In conclusion, ERα in osteocytes regulates trabecular bone formation and thereby trabecular bone volume in male mice but it is dispensable for the trabecular bone in female mice and the cortical bone in both genders. We propose that the physiological trabecular bone-sparing effect of estrogen is mediated via ERα in osteocytes in males, but via ERα in osteoclasts in females.Bone mass is maintained by highly regulated processes involving osteoblastic bone formation and osteoclastic bone resorption. Estrogen is the major sex hormone involved in the regulation of bone mass in females and several studies demonstrate that estrogen is also of importance for the male skeleton (1–6). The biological effects of estradiol (E2) are mainly mediated by the nuclear estrogen receptors (ERs), ERα encoded by the Esr1 gene, and ERβ encoded by the Esr2 gene. The bone-sparing effect of estrogen in both males and females is primarily mediated via ERα (6–8), although the effect of ERα activation in bone might be slightly modulated by ERβ in female mice (9–12).Two studies using different strategies for the inactivation of ERα in osteoclasts have determined the role of ERα in osteoclasts for the bone-sparing effect of estrogen (13, 14). Nakamura et al. used Ctsk (Cathepsin K)-Cre mice to inactivate ERα in mature osteoclasts, resulting in trabecular bone loss caused by increased bone resorption in female but not male mice (14). In a separate study Martin-Millan et al. inactivated ERα in monocytes/osteoclast precursors using LysM-Cre mice and found that estrogen attenuates osteoclast generation and life span via cell-autonomous effects and that ERα in osteoclasts mediates the protective effect of estrogens on trabecular but not cortical bone in female mice (13). Collectively, these two studies clearly demonstrated that ERα in osteoclasts is crucial for trabecular bone in females, but it is dispensable for trabecular bone in male mice and for cortical bone in both males and females. However, not only osteoclasts but also osteoblasts/osteocytes express ERs (15–17). Several in vitro studies have suggested that ERα in osteoblasts/osteocytes is of importance for the regulation of bone metabolism, but this has not yet been demonstrated in vivo (2, 16, 18).It is well established that mechanical loading is a major regulator of cortical bone dimensions (19, 20). Previous studies have demonstrated that female but not male mice with ERα inactivation display reduced cortical osteogenic bone response to mechanical loading (19, 21–24). In addition, we recently showed that ERα is required for the cortical osteogenic response to mechanical loading in a ligand-independent manner, involving activation function-1 but not activation function-2 in ERα in female mice (20). The primary ERα target cell for this role of ERα in the osteogenic bone response to mechanical loading is not yet characterized, but a plausible candidate is the osteocytes, which are the mechanosensors in bone.Because ERα in osteoclasts is required for the bone-sparing effect of estrogen specifically in trabecular bone in female mice, we hypothesized that ERα in osteocytes might be crucial for trabecular bone in male mice, for cortical bone in both genders, and for the cortical osteogenic response to mechanical loading in female mice (13, 14). Therefore, dentin matrix protein (Dmp)1-Cre [Tg(Dmp1-cre)1Jqfe] mice were crossed with ERα^flox/flox (Esr1^tm1.1Gust) mice to generate mice lacking ERα protein expression specifically in osteocytes (Dmp1-ERα^−/−). The main finding from this in vivo study shows that ERα in osteocytes is important for trabecular bone formation in male mice.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling

Synthetic matrices emulating the physicochemical properties of tissue-specific ECMs are being developed at a rapid pace to regulate stem cell fate. Biomaterials containing calcium phosphate (CaP) moieties have been shown to support osteogenic differentiation of stem and progenitor cells and bone tissue formation. By using a mineralized synthetic matrix mimicking a CaP-rich bone microenvironment, we examine a molecular mechanism through which CaP minerals induce osteogenesis of human mesenchymal stem cells with an emphasis on phosphate metabolism. Our studies show that extracellular phosphate uptake through solute carrier family 20 (phosphate transporter), member 1 (SLC20a1) supports osteogenic differentiation of human mesenchymal stem cells via adenosine, an ATP metabolite, which acts as an autocrine/paracrine signaling molecule through A2b adenosine receptor. Perturbation of SLC20a1 abrogates osteogenic differentiation by decreasing intramitochondrial phosphate and ATP synthesis. Collectively, this study offers the demonstration of a previously unknown mechanism for the beneficial role of CaP biomaterials in bone repair and the role of phosphate ions in bone physiology and regeneration. These findings also begin to shed light on the role of ATP metabolism in bone homeostasis, which may be exploited to treat bone metabolic diseases.Harnessing the ability of adult stem cells to differentiate and contribute to tissue repair has enormous potential for wound healing, tissue regeneration, and restoration of organ functionality. However, controlling the fate of transplanted and/or endogenous progenitor cells to treat compromised tissues and organs remains a significant challenge (1, 2). Studies have shown that biomaterials recapitulating various physicochemical cues of the native tissue can be used to direct stem cell differentiation (3–9). Biomaterials-assisted transplantation of stem cells provides a promising approach to deliver cells to the targeted site and direct their differentiation to functional tissues. We and others have shown that biomaterials containing calcium phosphate (CaP) moieties, a major constituent of native bone tissue, can promote osteogenic differentiation of progenitor and stem cells and can facilitate in vivo bone tissue formation (10–20). However, to use CaP biomaterials efficiently for bone tissue repair, it is of paramount importance to understand the molecular mechanisms underlying the osteogenicity (osteogenic differentiation of progenitor cells in the absence of any exogenous chemical or biological osteogenic-inducing factors) and osteoinductivity (de novo bone growth in vivo even in locations where there is no vital bone) of a CaP mineral environment.The osteogenicity and osteoinductivity of CaP minerals have been attributed to different factors, such as the ability of CaP to modulate extracellular calcium (Ca²⁺) and phosphate ions and the adsorption and release of osteoinductive growth factors like bone morphogenic proteins (BMPs) (18, 21–24). This is further supported by findings that exposure of osteoblasts and progenitor cells to Ca²⁺- or -rich medium promotes their osteogenic differentiation (25–27). Additionally, it has been shown that among various CaP materials, the ones that dissociate easily to Ca²⁺ and contribute to better bone healing (13, 21). Despite the large number of studies demonstrating the potential role of CaP minerals and Ca²⁺ and on osteogenic differentiation of osteoblasts and progenitor cells, the molecular mechanism through which these ions regulate osteogenic commitment of stem cells remains largely unknown. Recent studies have shown that influx of extracellular Ca²⁺ through L-type calcium channels promotes osteogenic differentiation of osteoprogenitor cells (28). However, very little is known about the mechanism through which supports osteogenesis. During skeletal growth and bone remodeling, plays an important role in apatite formation (29, 30). In addition to osteoblasts and progenitor cells, studies have shown that exposure to alters the cell phenotype of nonskeletal tissues, such as human vascular smooth muscle cells, into osteogenic-like cells (31, 32). Central to phosphate metabolism is solute carrier family 20 (phosphate transporter), member 1 (SLC20a1, or PiT-1), a sodium-phosphate symporter that transports ions from the extracellular milieu into the cytoplasm and plays a key role in mineralization of both vascular smooth muscle cells and osteoblasts (33, 34).Here, we unravel a previously unknown mechanism, centered on phosphate metabolism, through which the CaP-rich mineral environment promotes osteogenic differentiation of human mesenchymal stem cells (hMSCs) by using an engineered matrix containing CaP moieties. Our studies show that the extracellular plays an important role in promoting osteogenic differentiation of hMSCs by regulating intramitochondrial phosphate content and ATP synthesis. ATP is then secreted and metabolized into adenosine, which promotes osteogenic differentiation of hMSCs via A2b adenosine receptors.     

From the Cover: PNAS Plus: Functional screen identifies kinases driving prostate cancer visceral and bone metastasis

Mutationally activated kinases play an important role in the progression and metastasis of many cancers. Despite numerous oncogenic alterations implicated in metastatic prostate cancer, mutations of kinases are rare. Several lines of evidence suggest that nonmutated kinases and their pathways are involved in prostate cancer progression, but few kinases have been mechanistically linked to metastasis. Using a mass spectrometry-based phosphoproteomics dataset in concert with gene expression analysis, we selected over 100 kinases potentially implicated in human metastatic prostate cancer for functional evaluation. A primary in vivo screen based on overexpression of candidate kinases in murine prostate cells identified 20 wild-type kinases that promote metastasis. We queried these 20 kinases in a secondary in vivo screen using human prostate cells. Strikingly, all three RAF family members, MERTK, and NTRK2 drove the formation of bone and visceral metastasis confirmed by positron-emission tomography combined with computed tomography imaging and histology. Immunohistochemistry of tissue microarrays indicated that these kinases are highly expressed in human metastatic castration-resistant prostate cancer tissues. Our functional studies reveal the strong capability of select wild-type protein kinases to drive critical steps of the metastatic cascade, and implicate these kinases in possible therapeutic intervention.Metastatic prostate cancer is responsible for the deaths of ∼30,000 men in the United States each year (1, 2). Ninety percent of patients develop bone metastases, and other major sites of metastases include lymph nodes, liver, adrenal glands, and lung (3). First-line treatments for metastatic disease are androgen deprivation therapies that block androgen synthesis or signaling through the androgen receptor (AR) (2). Inevitably, metastatic prostate cancer becomes resistant to androgen blockade. Second-line treatments such as chemotherapy (docetaxel, cabazitaxel) and radiation only extend survival 2–4 mo (4, 5).Identifying new therapeutic targets for metastatic prostate cancer has proven difficult. Exome and whole-genome sequencing of human metastatic prostate cancer tissues have found frequent mutations and/or chromosomal aberrations in numerous genes, including AR, TP53, PTEN, BRCA2, and MYC (6–11). The precise functional contribution of these genes to prostate cancer metastasis remains unknown. Genomic and phosphoproteomic analyses have also revealed that metastatic prostate cancer is molecularly heterogeneous, which has complicated the search for common therapeutic targets (12). Few murine models of prostate cancer develop metastases. Mice having prostate-specific homozygous deletions in SMAD4 and PTEN or expression of mutant KRAS develop metastases in visceral organs but rarely in bone (13–15).Targeting genetically altered constitutively active protein kinases such as BCR-ABL in chronic myelogenous leukemia and BRAF^V600E in melanoma has led to dramatic clinical responses (16). Although numerous oncogenic alterations have been identified in prostate cancer, DNA amplifications, translocations, or other mutations resulting in constitutive activity of kinases are rare (6, 9, 17). Genome sequencing of metastatic prostate cancer tissues from >150 patients found translocations involving the kinases BRAF and CRAF in <1% of patients (8, 18). Although uncommon, these genomic aberrations cause enhanced BRAF and CRAF kinase activity and suggest that kinase-driven pathways can be crucial in prostate cancer. Multiple lines of evidence indicate that nonmutated kinases may contribute to prostate cancer progression, castration resistance, and metastasis. SRC kinase synergizes with AR to drive the progression of early-stage prostatic intraepithelial neoplasia to advanced adenocarcinoma (19). SRC, BMX, and TNK2 kinases promote castration resistance by phosphorylating and stabilizing AR (20–22). Moreover, FGFR1, AKT1, and EGFR kinases activate pathways in prostate cancer cells to drive epithelial-to-mesenchymal transition and angiogenesis, both of which are key steps in metastasis (23–25). Despite the strong evidence implicating kinases in advanced prostate cancer, a systematic analysis of the functional role of kinases in prostate cancer metastasis has been lacking.Metastasis of epithelial-derived cancers encompasses a complex cascade of steps, including (i) migration and invasion through surrounding stroma/basement membrane, (ii) intravasation and survival in circulation/lymphatics, (iii) extravasation through the vasculature, and (iv) survival and growth at a secondary site (26). With the exception of genetically engineered mouse models, no single experimental assay can model all steps of the metastatic cascade. As a result, most screens for genes involved in metastasis have focused on testing one step of the cascade. The migration/invasion step of metastasis is commonly interrogated in vitro by determining the ability of cells to invade through small pores in a membrane (27–29). Genes that function in other steps, or those dependent on the in vivo microenvironment to promote metastasis, are likely to be overlooked in these screens.Multiple groups have performed in vivo screens for regulators of metastasis by manipulating cell lines in vitro with shRNA libraries or using genome editing techniques, and injecting cells either subcutaneously or into the tail vein of mice (30, 31). These methods are advantageous, because they interrogate multiple steps of the metastatic cascade (survival in circulation, extravasation, and colonization and growth at a secondary site) in a physiologically relevant environment. However, the majority of in vivo screens conducted so far have been based on loss-of-function genetics. These screens are limited to inhibiting the function of proteins expressed by a particular cell line. Using a gain-of-function in vivo screen, we sought to identify kinases that activate pathways leading to prostate cancer metastasis.     

The WNT receptor FZD7 is required for maintenance of the pluripotent state in human embryonic stem cells

WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/β-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/β-catenin signaling through FZD7 to maintain an undifferentiated phenotype.Control of stem cell self-renewal is critical to the development of multicellular life; however, our understanding of the molecular machinery regulating this process remains superficial. Several studies have demonstrated that the WNT/β-catenin signaling pathway is a critical regulator of stem cell self-renewal, and the hypothesis that WNT primarily acts to maintain stem cells in an undifferentiated state has garnered significant support (reviewed in refs. 1–4). This paradigm is especially apparent in various adult stem cell populations, such as in skin, intestine, and blood, where WNT/β-catenin signaling is essential for proper tissue homeostasis.The role of WNT signaling in embryonic stem cells has been more controversial. In mouse embryonic stem cells, WNT/β-catenin signaling is active, and its inhibition shifts cells into an epiblast-like state (5–9). In contrast, in human embryonic stem cells (hESCs), which more closely resemble mouse epiblast stem cells than mouse embryonic stem cells (10, 11), WNT/β-catenin signaling is largely inactive, and ectopic stimulation of the pathway shifts them toward mesendodermal fates (12–14).Confounding the analysis of the role of WNT signaling in pluripotent stem cells is the large number of WNT ligands (the mammalian genome contains 19 Wnt genes) and WNT receptors encoded by the FZD gene family (the mammalian genome contains 10 Fzd genes), some of which may be acting redundantly. Furthermore, relatively little is known about the specificities of individual WNTs for individual receptors. Here we describe a set of experiments that demonstrate the presence of an endogenous WNT-FZD signaling loop that mediates a self-renewal signal in hESCs.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.     

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl^{PF4cre/PF4cre}). Mpl^{PF4cre/PF4cre} mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.Thrombopoietin (TPO) is the principal hematopoietic cytokine that regulates platelet production at steady state and is required for rapid responses to platelet loss. TPO acts by binding to a specific cell surface receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), leading to receptor dimerization, activation of intracellular signal transduction pathways, and responses of target cells. Mice lacking TPO or Mpl are severely thrombocytopenic and deficient in megakaryocytes and their progenitor cells, a phenotype consistent with a role for TPO in maintaining appropriate megakaryocyte numbers in vivo. In addition to its role in megakaryopoiesis, TPO is also an indispensible regulator of hematopoietic stem cells (HSC), essential for maintenance of quiescence and self-renewal (1).TPO is produced primarily in the liver (2) and upon binding to the Mpl receptor on target cells, is internalized and degraded. The prevailing model posits that circulating TPO concentration is inversely proportional to the “Mpl mass” contributed by the total number of megakaryocytes and platelets. In normal individuals, this model describes an effective feedback system to regulate TPO-driven megakaryocyte and platelet production according to need. The reciprocal relationship between platelet number and circulating TPO level is clearly evident in bone marrow transplant patients (1), and the key role of the TPO receptor is illustrated by the elevated circulating TPO in Mpl^−/− mice (3) and the modest elevation of platelet counts in transgenic mice expressing low levels of Mpl (4, 5). However, the relationship between circulating TPO concentration and peripheral platelet counts is not always conserved in pathological states of thrombocytosis and thrombocytopenia (6–9), suggesting that a simple relationship among megakaryocyte and platelet Mpl mass, circulating TPO concentration, and the degree of stimulation of megakaryopoiesis may not always hold.Whereas expression of Mpl on megakaryocytes and platelets contributes to regulation of available TPO, the role of direct TPO stimulation of megakaryocytes for effective platelet production is unclear. Administration of TPO in vivo or stimulation of bone marrow in vitro elevates megakaryocyte numbers and increases mean DNA ploidy (10, 11), and the thrombocytopenia in TPO^−/− mice is accompanied by reduced megakaryocyte ploidy (12). However, although exposure of megakaryocytes to TPO stimulates intracellular signaling (13), in vitro studies suggesting direct action of TPO on megakaryocytes to increase DNA ploidy, promote cytoplasmic maturation, or to stimulate proplatelet production (14–16) are balanced by reports that TPO is dispensable for these megakaryocyte functions (15, 17–19).To comprehensively define Mpl-expressing stem and progenitor cell populations in vivo and resolve the specific requirements for Mpl expression in megakaryocytes and platelets for platelet production and feedback control of TPO levels, we generated a mouse strain in which green fluorescent protein (GFP) is expressed from the Mpl locus and, when crossed to Platelet Factor 4(PF4)cre transgenic mice (20), specifically lacks Mpl expression in megakaryocytes and platelets.